• Extracellular nuclear proteins H4 and HMGB1 are highly proinflammatory cytokines.• Inorganic polyP dramatically amplifies proinflammatory responses of H4 and HMGB1 through the RAGE and P2Y 1 receptors.

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the ␥-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGFlike domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K D ) of ϳ7 nM. However, the apparent K D for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes ϳ6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes. Protein Z (PZ)2 is a vitamin K-dependent coagulation glycoprotein, which has a genetic organization that is identical to that of factor Xa (fXa) and other similar vitamin K-dependent coagulation factors (1, 2). Thus, PZ has an N-terminal ␥-carboxyglutamic acid (Gla) domain that is followed by two epidermal growth factor (EGF)-like domains (light chain homologue) and a C-terminal pseudo catalytic domain (heavy chain homologue) (1, 2). Unlike fXa and other vitamin K-dependent coagulation proteases, two of the catalytic triad residues of the C-terminal domain have not been conserved in the homologous regions of PZ (1, 2). Thus, PZ has no catalytic function, but instead it binds to PZ-dependent protease inhibitor (ZPI) with a high affinity, thereby promoting the ZPI inhibition of fXa in the presence of PC/PS vesicles and Ca 2ϩ by approximately three orders of magnitude (3-5). ZPI is a 72-kDa serpin with a plasma concentration of 2.6 -2.9 g/ml that appears to interact with the active site pocket of fXa by a covalent mechanism similar to that observed with antithrombin and most other inhibitory serpins (4 -6). However, unlike antithrombin, which can inhibit all coagulation proteases, ZPI is a specific inhibitor of fXa and factor XIa (4, 7, 8), though recent results have indicated that it may also inhibit factor IXa (9). ZPI may play a critical role in the regulation of the coagulation cascade because its deficiency is associated with venous thromboembolic diseases (10, 11). ZPI by itself is a poor inhibitor of fXa, unless i...

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of metallonuclease mechanism. DNA association rate constants are about 100-fold higher in the presence of the catalytically nonsupportive Ca(II) vs. the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM2S and EM4S) support cleavage. Interestingly, the cleavage rate for EM2S is about 100-fold slower than that displayed by EM4S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.

FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56+/-1 A (1 A=0.1 nm) and 63+/-1 A for fXa-desEGF1 compared with 72+/-2 A and 75+/-1 A for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa2=2/3. In comparison, an L value of 95+/-6 A was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys(195) of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to Fl-FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface.